Leuconostoc mesenteroides Jonggajibkimchi subspecies CGMCC (China General Microbiological Culture Collection Center) No.30904 and application thereof

Abstract

The invention discloses a leuconostoc mesenteroides substrain, the preservation number of the strain is CGMCC (China General Microbiological Culture Collection Center) No.30904, the preservation date is June 7, 2024, the preservation classification is named as leuconostoc mesenteroides, and the preservation unit is the General Microbiological Culture Collection Center of the China Committee for Culture Collection of Microorganisms. The strain provided by the invention is harmless to animals, and has the remarkable effects of relieving lung tissue inflammatory infiltration caused by bronchial asthma, reducing release of serum IgE and Th2 type cell factors, inhibiting excessive lung macrophage replacement activation and improving allergic airway inflammation.

Description

Technical Field

[0001] The invention relates to a preserved strain of Leuconostoc mesenteroides jonggajibkimchii subspecies and application thereof, belonging to the field of microorganisms. Background Art

[0002] Bronchial asthma, abbreviated as asthma, is a heterogeneous chronic inflammatory disease. The typical characteristics of asthma are varying degrees of airflow limitation, accompanied by recurrent symptoms such as wheezing, shortness of breath, cough and chest tightness, which can lead to airway remodeling as the disease progresses. Asthma is one of the chronic diseases that seriously threaten public health worldwide. In recent years, epidemiological studies have confirmed that there are more than 358 million asthma patients worldwide, at least 250,000 people die from the disease each year, and the prevalence rate has shown a significant upward trend since 1990. According to the "2022 Global Asthma Report", asthma ranks 24th among the leading causes of disability worldwide and 34th among the leading causes of disease burden. 96% of asthma-related deaths and 84% of related disabilities worldwide occur in low- and middle-income countries (developing countries), which brings huge economic and medical burdens to asthma patients and society.

[0003] The heterogeneity of asthma is manifested in different causes, pathogenic mechanisms, clinical symptoms and severity. According to the latest Global Initiative for Asthma (GINA) asthma prevention and treatment guidelines, asthma can be divided into type 2 and non-type 2 types according to the presence or absence of type 2 (T2) inflammation. This classification method represents different molecular mechanisms and is called endotype. Type 2 asthma is the most common, accounting for 64%-73% of all asthma patients, and is mainly manifested by an increase in eosinophils and a significant increase in T helper 2 cell (Th2) type cytokines interleukin-4 (IL-4), IL-5 and IL-13. Th2 cytokines promote eosinophilia and lead to the activation of B cells into plasma cells, releasing a large amount of specific immunoglobulin E (IgE), which promotes mast cell degranulation, releases multiple factors such as leukotrienes and histamine, and causes airway smooth muscle contraction and spasm, resulting in asthma symptoms. This type of asthma usually responds well to glucocorticoids or new biological agents, but there are certain side effects. Non-type 2 asthma is more common in obesity-related asthma, neutrophilic asthma, and oligogranulocytic asthma, which is characterized by the infiltration of Th1 and Th17 cells and neutrophils, the large production of NLR Family, Pyrin Domain Containing Protein 3 (NLRP3), and type I interferon, IL-1β, and IL-17. This type of asthma usually responds poorly to drugs such as steroids. Therefore, it is very necessary to find anti-asthma interventions without side effects.

[0004] The development of immune function and susceptibility to disease are strongly influenced by a variety of resident microorganisms or microbiota. Therefore, many studies have attempted to guide the development of immune function by administering specific microorganisms or probiotics, thereby potentially preventing or treating allergic diseases. For example, Irina Spacova et al. found that the probiotic Lactobacillus rhamnosus can prevent the occurrence of airway hyperresponsiveness, inhibit eosinophilia, and prevent the deterioration of airway function in allergic asthma; AnnaMarlene Schmid et al. reported that the probiotic Escherichia coli O83 can release outer membrane vesicles, inhibit airway hyperresponsiveness, reduce airway eosinophils, Th2-type cytokine production and mucus secretion, and play a protective role in asthma. However, there are not many studies in this field at present, and the number and types of probiotic strains reported to have anti-asthma function are limited, and further exploration is still needed.

[0005] Lactic acid bacteria play an important role in the healthy development of life. They can maintain the homeostasis of intestinal microorganisms through various pathways, produce a variety of bioactive compounds such as peptides, bacteriocins, proteases, lipases and lactic acid during fermentation, and improve the immunity and disease resistance of animals. Leuconostoc mesenteroides ) is a type of facultative anaerobic Gram-positive bacteria, an important species of the genus Leuconostoc, and is listed in the "List of Bacteria that Can Be Used in Food". It has antibacterial, antioxidant and metabolic regulation functions. Gao Kan et al. found that oral administration of Leuconostoc mesenteroides WHH1141 can upregulate the intestinal microbial diversity and short-chain fatty acid levels of mice, and alleviate food allergies induced by ovalbumin (OVA), suggesting that Leuconostoc mesenteroides has the function of regulating immunity.

[0006] Macrophages can be divided into classically activated macrophages (M1) and alternatively activated macrophages (M2) according to their activation status. M1 macrophages release pro-inflammatory factors such as tumor necrosis factor-α (TNF-α) and IL-1β, aggravating the inflammatory response; while M2 macrophages are divided into three subtypes: M2a, M2b, and M2c, which mainly produce anti-inflammatory cytokines such as IL-10 and have strong phagocytic function. The exact role of M2 macrophages in asthma has not been fully clarified, but it is known that they promote Th2 immune response, which is particularly important in allergic asthma. The number of M2 macrophages in asthma patients is associated with the severity of asthma symptoms and promotes Th2 inflammatory response in allergic asthma. In children with asthma, different subsets of macrophages in the peripheral blood play different roles in the evolution and prognosis of asthma. Studies have shown that M1 macrophages in the peripheral blood of children with asthma are positively correlated with the severity of asthma, while M2 macrophages are negatively correlated.

[0007] The purpose of the present invention is to provide a probiotic that has the functions of reducing lung inflammatory cell infiltration, lowering lung Th2 cytokine and serum IgE levels, inhibiting excessive macrophage replacement activation in the lungs, and relieving allergic airway inflammation. Summary of the invention

[0008] Based on the above invention purpose, the present invention first provides a strain of Leuconostoc jonggajibkimchii subspecies MY2024 (hereinafter referred to as MY2024), the deposit number of the strain is CGMCC No.30904, the deposit date is June 7, 2024, and the deposit classification is named Leuconostoc mesenteroides Leuconostoc mesenteroidesThe strain is isolated from black bean fermentation liquid and is listed in the "List of Bacteria that can be used in food" issued by the National Health Commission.

[0009] In a preferred embodiment, the sequence of 16S rRNA of the strain is shown as SEQ ID NO.1.

[0010] Secondly, the present invention provides the use of the above strain in preparing drugs for treating or preventing allergic airway inflammation.

[0011] In a preferred embodiment, the allergic airway inflammation is asthma.

[0012] More preferably, the asthma is accompanied by an increase in serum IgE and / or Th2 cytokines in the airways.

[0013] More preferably, the asthma is accompanied by inflammatory cell infiltration, goblet cell hyperplasia, mucus production and collagen deposition in the airways.

[0014] More preferably, the asthma is accompanied by excessive enhancement of alternative activation of pulmonary macrophages.

[0015] Finally, the present invention provides a composition containing the above strains. The strains can be used in combination with other drug components to improve or promote the therapeutic effect of asthma, and other components include but are not limited to probiotics, chemical molecules, protein molecules, and nucleic acid molecules that have therapeutic effects on asthma.

[0016] In a preferred embodiment, the composition further contains a pharmaceutically acceptable carrier and / or excipient.

[0017] In a more preferred embodiment, the composition is prepared as capsules, lyophilized powders, suspensions or tablets.

[0018] The present invention is a strain of Leuconostoc mesenteroides with anti-asthma properties separated and purified from black bean fermentation liquid. Experiments have shown that the separated Leuconostoc mesenteroides subsp. jonggajibkimchii The MY2024 strain is harmless to animals and has been confirmed to have the function of improving asthma through animal experiments. Leuconostoc mesenteroides subsp. jonggajibkimchii The MY2024 strain can effectively reduce the serum IgE level of asthma model mice, reduce the production of Th2 cytokines in the lungs, alleviate the infiltration of inflammatory cells in the lungs and the production of airway mucus, and reduce excessive macrophage alternative activation in the lungs, showing excellent application prospects in the preparation of asthma treatment and / or prevention drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Animal experimental protocols used in this invention; Figure 2 Oral administration of MY2024 can significantly reduce the number of lung cells in the OVA mouse model. The horizontal axis represents different animal treatment groups, and the vertical axis represents the number of cells. * indicates p<0.05, ** indicates p<0.01, and *** indicates p<0.001. Figure 3-Figure 6 Oral administration of MY2024 can significantly reduce the percentage of eosinophils and macrophages in the lungs of OVA mice models, but does not affect the percentage of neutrophils and monocytes. * indicates p<0.05, ** indicates p<0.01, and *** indicates p<0.001. Figure 3 Effects of oral administration of MY2024 on the percentage of eosinophils in mouse lung tissue; Figure 4 Effects of oral administration of MY2024 on the percentage of macrophages in mouse lung tissue; Figure 5 Effects of oral administration of MY2024 on the percentage of neutrophils in mouse lung tissue; Figure 6 Effects of oral administration of MY2024 on the percentage of monocytes in mouse lung tissue; Figure 7 It shows that oral administration of MY2024 can significantly reduce the serum IgE level of OVA mouse model. The horizontal axis represents different animal treatment groups, and the vertical axis represents serum IgE content. * indicates p<0.05, ** indicates p<0.01, and *** indicates p<0.001. Figure 8-Figure 10 It shows that oral administration of MY2024 can significantly reduce the levels of IL-4, IL-5 and IL-13 in the lung tissue of OVA mouse models. The horizontal axis represents different animal treatment groups, and the vertical axis represents the corresponding cytokine content. * indicates p<0.05, ** indicates p<0.01, and *** indicates p<0.001; Figure 8 Effects of oral administration of MY2024 on IL-4 levels in mouse lung tissue; Fig. 9 Effects of oral administration of MY2024 on IL-5 levels in mouse lung tissue; Fig.10 Effects of oral administration of MY2024 on IL-13 levels in mouse lung tissue; Fig.11 Shows the results of H&E staining comparison of lung tissue sections of mice in each group after oral administration of MY2024 (I); Fig.12 Shows the results of H&E staining comparison of lung tissue sections of mice in each group after oral administration of MY2024 (B); Fig.13Shows the results of H&E staining comparison of lung tissue sections of mice in each group after oral administration of MY2024 (III); Fig.14 Shows the comparison results of PAS staining of lung tissue sections of mice in each group after oral administration of MY2024 (I); Fig.15 Shows the comparison results of PAS staining of lung tissue sections of mice in each group after oral administration of MY2024 (B); Fig.16 Shows the comparison results of PAS staining of lung tissue sections of mice in each group after oral administration of MY2024 (I); Fig.17 Shows the comparison results of MASSON staining of lung tissue sections of mice in each group after oral administration of MY2024 (I); Fig.18 Shows the comparison results of MASSON staining of lung tissue sections of mice in each group after oral administration of MY2024 (B); Fig.19 Shows the comparison results of MASSON staining of lung tissue sections of mice in each group after oral administration of MY2024 (III); Figure 20-23 Oral administration of MY2024 significantly reduced the transcription levels of alternative activation marker genes in lung tissue macrophages in the OVA mouse model; Fig. 20 Effects of oral administration of MY2024 on the relative gene expression of CD206 in mouse lung tissue; Fig.21 Effects of oral administration of MY2024 on the relative gene expression of Arg1 in mouse lung tissue; Fig. 22 Effects of oral administration of MY2024 on the relative gene expression of Fizz1 in mouse lung tissue; Fig.23 Effects of oral administration of MY2024 on the relative gene expression of Ym1 in mouse lung tissue; Fig.24 It showed that oral administration of MY2024 could significantly reduce the protein level of Arg1, a marker gene for alternative activation of macrophages in the lung tissue of OVA mouse models. DETAILED DESCRIPTION

[0020] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of protection defined by the claims of the present invention.

[0021] Example 1. Isolation, preservation and identification of Leuconostoc mesenteroides MY2024 1.1 Isolation of Leuconostoc mesenteroides MY2024 (1) Take 100 μL of black bean fermentation liquid sample and add it to an EP tube pre-filled with 900 μL of sterile PBS. Dilute the sample in sequence until the concentration of the sample extract is diluted to 10 -6 times; (2) Take 100 μL of samples with different dilutions and spread them on MRS solid culture medium and place it in the incubator; (3) Culture at 37°C, 0.5% CO2 for 48 h; (4) Remove the culture dish and use a sterile inoculation loop to pick colonies with different morphological characteristics. Transfer them to new MRS medium for purification. Culture anaerobically at 37°C for 48 h. Transfer the culture medium three times in a row and culture the purified strains in liquid MRS at pH 3.5. Select strains with excellent acid-resistant growth for use in experiments or frozen storage.

[0022] 1.2 Strain preservation This laboratory uses MRS medium containing 25% glycerol as the bacterial preservation solution for frozen preservation of bacterial strains. The method is as follows: (1) Sterilize a 2 mL sterilization tube at 121°C for 15 min before use; (2) After the bacteria have been transferred to MRS solid medium three times, add 1.5 mL of sterile bacterial preservation solution to the culture dish; (3) Use an L-rod to scrape the culture dish to ensure that the colonies are fully integrated into the bacterial preservation solution; (4) Transfer the bacterial solution to a bacterial preservation tube, mix well, and store at -80℃.

[0023] 1.3 Observation of colony appearance and bacterial morphology Leuconostoc mesenteroides subspecies jonggajibkimchii is a facultative anaerobic bacterium that grows well under anaerobic conditions. Its colonies are round, milky white, smooth, with neat edges and are opaque. Under a microscope, it is a Gram-positive coccus.

[0024] 1.4 Extraction of total bacterial DNA A single colony was inoculated on MRS solid medium and cultured anaerobically overnight at 37°C. DNA was extracted according to the instructions of the bacterial genomic DNA extraction kit (Nanjing Novozyme FastPure® DNA Extraction Kit).

[0025] 1.5 Accurately identify strains by comparing ANI and DDH with model strains The genome of the strain was extracted and draft sequenced. The genome correlation analysis (digital DNA-DNA hybridization (dDDH; dDDH < 70%) and average nucleotide identity (Average Nucleotide Identity, ANI; ANI < 95%)) was performed by comparison with the model strain to further accurately identify the strain. Both methods are the gold standard for determining prokaryotic species and are completed online (dDDH, http: / / ggdc.dsmz.de; ANI, http: / / enve-omics.ce. gatech.edu / ani / ).

[0026] 1.5.1 Genome extraction and draft submission This study used the DNA extraction kit produced by Nanjing Novozyme Biotechnology Co., Ltd. for gene extraction. The specific operation steps are as follows: (1) Since the bacteria are Gram-positive, lysozyme solution needs to be added to the bacterial precipitate and treated at 37°C for 2 h to ensure that the cell wall is fully destroyed.

[0027] (2) Add 20 μl of proteinase K solution to the above mixture and shake to remove protein impurities; then add 200 μl of buffer BCL and shake to mix; then incubate in a 55°C water bath for 10 min, invert and mix to ensure that there are no impurities precipitated in the solution.

[0028] (3) Add 150 μl of anhydrous ethanol and shake to mix. Flocculent precipitate will be generated during this process. Transfer all the liquid and floccules in the EP tube to the adsorption column FastPure gDNA Mini Columns II, centrifuge at 12000 rpm for 30 seconds, and discard the waste liquid.

[0029] (4) Place the adsorption column in the waste liquid collection tube, add 500 μl of buffer WA to the adsorption column, centrifuge at 12000 rpm for 1 min, and discard the waste liquid.

[0030] (5) Add 600 μl of WB buffer, centrifuge at 12,000 rpm for 30 s, discard the waste liquid, put it into a new collection tube, and centrifuge at 12,000 rpm for 2 min. Perform this step twice to ensure that the DNA is clean.

[0031] (6) Place the adsorption column in a collection tube, open the lid and leave it at room temperature for 3 to 5 minutes to dry. This is to allow the remaining rinse solution (mainly ethanol) in the adsorption column to evaporate.

[0032] (7) Transfer the adsorption column to a new EP tube, add 200 μl of Elution Buffer (preheated to 55°C) to the adsorption column membrane, place at room temperature for 2-5 minutes, and centrifuge at 12,000 rpm for 1 minute to obtain pure bacterial genomic DNA. Transport the cold chain to Beijing Tianyi Huiyuan Biotechnology Co., Ltd. to complete the draft sequencing of the genome.

[0033] 1.5.2 Genome correlation analysis (1) Basic characteristics of the genome The draft results showed that the genome size of the MY2024 strain was 198,873 bp and the GC content was 42.36%.

[0034] (2) dDDH and ANI Selection of a type strain of Leuconostoc mesenteroides subsp. jonggajibkimchii Leuconostoc mesenteroides subsp. mesenteroides The dDDH and ANI values ​​of ATCC 8293 and MY2024 were compared. The results showed that the dDDH and ANI values ​​between the model strain ATCC 8293 and MY2024 were 86.20% and 98.87%, respectively, supporting that the two strains were the same bacterial species.

[0035] (3) 16S RNA sequence determination The single colony of the bacteria was picked and added to 10 mL of MRS liquid culture medium, shaken at 37°C overnight, and transported by cold chain to Beijing Tianyi Huiyuan Biotechnology Co., Ltd. to complete 16S RNA sequence determination. After sequence determination, the 16S RNA sequence of the MY2024 strain is shown in SEQ ID NO.1.

[0036] The preservation information of this strain is as follows: the strain preservation number is CGMCC No.30904, the preservation date is June 7, 2024, and the preservation classification is named Enterococcus mesenteroides Leuconostoc mesenteroides The depository is the General Microbiology Center of China Culture Collection Administration, whose address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101, Tel: 8610-64807355.

[0037] Example 2 Evaluation of the anti-asthma function of Leuconostoc mesenteroides MY2024 strain 2.1 Sample: Activated culture of Leuconostoc mesenteroides MY2024 strain in logarithmic growth phase.

[0038] 2.2 Experimental animals and OVA animal model construction 5-week-old female BALB / c mice were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd., with a weight range of 13-15 g. The license number is SCXK (Beijing) 2064-0006. The breeding location is the Laboratory Animal Center of the Chinese Center for Disease Control and Prevention, license number SYXK (Beijing) 2017-0021, the breeding level is a barrier environment, 12-h light / dark cycle, and mice have free access to water and food. All animal experiments were carried out in accordance with the ethical guidelines for experimental animal welfare of the Laboratory Animal Center of the Chinese Center for Disease Control and Prevention (approval number: 2023-025).

[0039] According to the experimental requirements, the mice were randomly divided into three groups: normal group (PBS group), model group (OVA group) and probiotic intervention group (MY2024), with 8 mice in each group. After one week of adaptation, the mice in the PBS group and OVA group were gavaged with 200 μL PBS every day from 5 days before infection (day -5) to the 16th day after infection (the day of infection was recorded as day 0), and the mice in the MY2024 group were gavaged with 200 μL MY2024 bacterial suspension (1×10 9 CFU / mouse). On days 0 and 7, mice in the OVA group and MY2024 group were sensitized by intraperitoneal injection of 100 μL ovalbumin suspension (prepared on the same day, containing 100ug OVA), and mice in the PBS group were injected with an equal volume of PBS. On days 14-16, mice in the OVA group and MY2024 group were given nebulized inhalation of 1% OVA solution to stimulate asthma, each time for 20 min, while mice in the PBS group were given nebulized inhalation of normal saline instead.

[0040] 2.3 Sample collection: Mice were killed 24 hours after the last nebulization challenge, and serum and lung tissues were collected.

[0041] (1) Blood sample collection Blood was collected from the eyeball. The mouse whiskers were trimmed to avoid hemolysis; the eyeballs of the mice were quickly plucked with curved forceps, and the whole blood of the mice was collected in a 1.5 mL sterile EP tube. After the collection was completed, the blood was placed in a 37 ℃ water bath for 1 h, centrifuged at 3500 rpm for 15 min, and the serum was packaged and stored at -80 ℃ after being marked. Pay attention to avoid hemolysis during the collection process and avoid repeated freezing and thawing.

[0042] (2) Lung tissue: The chest was dissected to expose the heart and both lungs. The left lung was removed and the surrounding excess tissue was removed. Tissue of approximately 1 cm × 1 cm × 1 cm in size was obtained from the middle lobe of the left lung and fixed with 4% paraformaldehyde for pathological sectioning. The right lung was stored at -80°C. All mouse carcasses were sent to the Experimental Animal Center for recovery and unified treatment.

[0043] 2.4 Detection method: 2.4.1 Detection of serum IgE content: Use a commercial kit (IgE Mouse Uncoated ELISA Kit, invitrogen, Cat#88-50460) for detection according to the instructions.

[0044] 2.4.2 Detection of Th2 cytokines (IL-4, IL-5 and IL-13) in lung homogenate supernatant: Commercial kits were used for detection according to the instructions, including IL-4 (Mouse IL-4 Uncoated ELISA, invitrogen, Cat#88-7044), IL-5 (Mouse IL-5 Uncoated ELISA, invitrogen, Cat#88-7054), and IL-13 (Mouse IL-13 Uncoated ELISA, invitrogen, Cat#88-7137).

[0045] 2.4.3 Flow cytometry detection of cell ratio: (1) Kill the mice, open the chest cavity, expose the heart and lung tissue, remove the mouse lung tissue, and place it in pre-cooled RPMI1640 culture medium.

[0046] (2) After rinsing the lung tissue with PBS, place a 40 μm nylon mesh on a 6-well plate. Pipette 2 mL of RPMI 1640 and place it on the nylon mesh. Use a grinding rod to slowly grind the cells so that they pass through the filter mesh and flow into the 6-well plate. Collect the grinding solution into a 15 mL centrifuge tube and centrifuge at 1500 rpm for 5 min.

[0047] (3) Collect the supernatant and store at -80℃ for Th2 cytokine ELISA detection. Resuspend the cells with 2 mL of digestion solution (containing 2 mg / mL collagenase I), place the centrifuge tube in a 37℃ shaker at 200 rpm / min for 20 min, then add RPMI 1640 medium containing 10% FBS to terminate digestion, and centrifuge at 1500 rpm for 5 min.

[0048] (4) Discard the supernatant, add 2 mL of red blood cell lysis buffer, blow off quickly, let stand at room temperature for 5 min, then add 2 mL of 10% FBS RPMI 1640 to stop the process, and centrifuge at 1500 rpm for 5 min. Discard the supernatant, add 1 mL of PBS to resuspend the cells, take a portion to count the cells using a cell counter, and then centrifuge at 1500 rpm for 5 min.

[0049] (5) Adjust the cell concentration to 1×10 6cells / 100 μL cell suspension, anti-CD16 / 32 was added to block Fc receptors, and incubated on ice in the dark for 10 min. Then anti-CD11b-APC / Cyanine7 (Biolegend, Cat#101226), anti-F4 / 80-PE / Cyanine7 (Biolegend, Cat#123114), anti-CD45-AF700 (Biolegend, Cat#103128), anti-SiglecF-BV421 (Biolegend, Cat#155509), anti-Ly6G-PE (Biolegend, Cat#127608), and anti-Ly6C-Percp / Cyanine5.5 (Biolegend, Cat#128028) were added and incubated on ice in the dark for 30 min.

[0050] (6) Add 1 mL of PBS and centrifuge at 400 g for 5 min. Repeat twice.

[0051] (7) Add 150 μL Fixation Buffer (Biolegend, Cat#420801) and incubate at room temperature in the dark for 20 min. Then add 1 mL 1× Permeabilization Wash Buffer (Biolegend, Cat#421002) and centrifuge at 400 g for 5 min. Discard the supernatant, add 1 mL PBS to resuspend, centrifuge and discard the supernatant. Add 300 μL PBS to resuspend, sieve and transfer to a flow tube for loading. Fluorescence intensity is displayed as a percentage, and the results are analyzed using FlowJo software version 10.8.1.

[0052] 2.4.4 Pulmonary pathology: The tissues were sealed with 4% paraformaldehyde for 24 h in advance. Pathological section preparation, H&E staining, PAS staining, MASSON staining, and section scanning were all completed by Wuhan Saiweier Biotechnology Co., Ltd. CaseViewer (version 2.3.0) was used for reading.

[0053] 2.4.5 Tissue RNA extraction and real-time quantitative PCR detection: The tissue was placed in a grinding tube containing 1 mL of Trizol and one grinding bead was added to each tube. The grinding tube was then placed in a grinding module precooled at -80°C for 2 h and ground on the machine. After grinding, 200 μL of chloroform was added to each tube and shaken to mix, and then placed on ice for 20 min. After completion, the upper layer (aqueous phase) was taken by centrifugation at 12,000 rpm at 4°C for 20 min, and 500 μL of isopropanol was added to mix by inversion. After incubation at room temperature for 10 min, the supernatant was discarded and the white precipitate was washed with 75% ethanol to obtain RNA, which was dissolved in 100 μL of enzyme-free water and the RNA concentration was determined. The reverse transcription reaction solution system (40 μL per tube) was prepared according to the reagent manufacturer's instructions, 500 ng of RNA was added to each tube, and reverse transcription was performed on the machine at 37°C for 15 min and 85°C for 5 min to obtain cDNA. The quantitative PCR reaction solution system was prepared according to the reagent manufacturer's instructions (20 μL per tube, containing 2 μL DNA template), and the quantitative PCR test was performed on the machine. The reaction conditions were: 95°C pre-denaturation for 30 seconds; then 95°C denaturation for 3 seconds, 60°C annealing for 30 seconds, and 40 cycles. The system collected the fluorescence signal at the end of each cycle. β-actin was used as the internal reference gene, and the relative expression of each target gene was calculated by the 2-ΔΔCt method.

[0054] Table 1. Primer sequences used for PCR

[0055] 2.4.6 Tissue protein extraction and Western blot detection: Place the tissue in a grinding tube containing 1 mL PBS and add one grinding bead to each tube. Then place the grinding tube in a grinding module pre-cooled at -80℃ for 2 hours and grind on the machine; after grinding, take 200 μL of homogenate and add an equal amount of RIPA protein lysis buffer (containing protease inhibitors), and mix thoroughly with a micropipette. After standing on ice for 30 minutes, centrifuge at 12000rpm at 4℃ for 10 minutes. Transfer the supernatant to a new 1.5 mL EP tube, retain 2 μL of each sample for protein quantification, and add 1 / 4 of its volume of 5× protein loading buffer to the remaining sample, and heat it in a 95℃ metal bath for 10 minutes. Centrifuge at 12000rpm at 4℃ for 5 minutes, collect the prepared protein samples, and freeze them at -80℃ for subsequent research. Use a commercial kit (Pierce TMBCA Protein Assay Kits, Cat#23227) was used for BCA protein quantification according to the instructions. Commercial precast gel (Lablead, Cat#P01015) was used. After adding electrophoresis buffer, 30 μg of sample protein was added to the spotting well, and electrophoresis was performed at 160 V constant voltage for about 40 min. 1× transfer buffer was used to activate the nitrocellulose membrane (NC membrane), and the protein was transferred to the NC membrane by wet transfer method at a constant current of 250 mA for 47 min. Then, 5% skim milk in 1× TBST was used to block at 37°C for 1 h, and the membrane was washed three times with 1× TBST for 5 min each time to remove the residual skim milk. The required bands were cut according to the protein marker and incubated with primary antibodies at 4°C overnight. The membrane was washed three times with 1× TBST the next day, and the bands were incubated with the corresponding HRP-labeled secondary antibody at 37°C for 1 h. Then, the membrane was washed three times with 1× TBST, and the expression of the corresponding protein was detected by electrochemiluminescence (ECL) method.

[0056] 2.5 Statistical analysis methods: All data were statistically analyzed using GraphPad Prism 9.1.0. Data are expressed as mean ± standard deviation (SD). Differences between groups were analyzed using one-way analysis of variance and Turkey test as post hoc multiple comparisons. Data that did not conform to normal distribution were logarithmically transformed or subjected to nonparametric analysis.

[0057] 2.6 Results To observe the effect of Leuconostoc mesenteroides MY2024 strain in relieving asthma, we used 5-week-old female BALB / c mice weighing 13-15 g, and induced asthma in mice by intraperitoneal injection of OVA sensitization + nebulization challenge. From 5 days before the first sensitization (day -5) to the last nebulization challenge (D16), the mice were gavaged with 200 μL of Leuconostoc mesenteroides MY2024 strain (1×10 9 CFU / mouse), and the model group and blank control group were gavaged with the same volume of PBS every day. The mice were killed 24 hours after nebulization stimulation, and the total number of lung cells, percentage of classified cells, serum IgE, lung Th2 cytokines, lung inflammatory cell infiltration, mucus secretion, and collagen deposition levels between the MY2024 group and the OVA group were compared to evaluate the anti-asthmatic effect of the MY2024 strain. The mRNA and protein expression levels of alternative activation gene markers of lung tissue macrophages of the MY2024 group and the OVA group were compared to explore the anti-asthmatic mechanism of the MY2024 strain.

[0058] (1) If Figure 1As shown, this study was divided into three groups, namely PBS group (normal group), OVA group (OVA sensitization challenge) and MY2024 group (OVA sensitization challenge and oral MY2024 intervention). The results showed: Figure 2 The results showed that oral administration of Leuconostoc mesenteroides MY2024 strain could significantly reduce the total number of lung cells in asthmatic mice (1.67±0.10×10 in the PBS group). 6 / mL, OVA group 3.98±0.25×10 6 / mL, MY2024 group 2.64±0.33×10 6 / mL).

[0059] Figure 3-Figure 6 The results showed that the proportion of eosinophils and macrophages decreased after oral administration of Leuconostoc mesenteroides MY2024, while there was no significant difference in the proportion of neutrophils and monocytes. Figure 3 The percentage of eosinophils in each group was shown as follows: 0.47±0.12 in the PBS group, 18.47±3.38 in the OVA group, and 6.36±2.13 in the MY2024 group; Figure 4 The percentage of macrophages in each group was shown as follows: 0.83±0.09 in the PBS group, 11.97±2.64 in the OVA group, and 4.86±1.08 in the MY2024 group; Figure 5 The percentage of neutrophils in each group was shown as follows: 5.17±1.16 in the PBS group, 4.98±1.04 in the OVA group, and 5.43±1.91 in the MY2024 group; Figure 6 The percentage of monocytes in each group was shown as follows: 5.80±1.29 in the PBS group, 6.01±0.98 in the OVA group, and 5.67±1.80 in the MY2024 group).

[0060] In addition, the strain can effectively reduce the serum IgE of asthmatic mice ( Figure 7 : PBS group 4.11±0.39 μg / mL, OVA group 52.4±10.02 μg / mL, MY2024 group 26.85±3.82 μg / mL). This strain can also effectively reduce the Th2 cytokine IL-4 in the lung ( Figure 8 : PBS group 49.72±5.68 pg / mL, OVA group 98.41±9.69 pg / mL, MY2024 group 66.75±6.51 pg / mL), IL-5 ( Fig. 9 : PBS group 198.30±2.62 pg / mL, OVA group 426.96±80.03 pg / mL, MY2024 group 266.39±25.25 pg / mL) and IL-13 ( Fig.10: 14.79±0.66 pg / mL in PBS group, 34.22±5.85 pg / mL in OVA group, and 19.80±1.77 pg / mL in MY2024 group).

[0061] (2) Pathological sections were taken from the left lung lobes of mice 24 hours after the last nebulization challenge to observe the pathological changes in the lung tissue of mice. The results of H&E staining showed that compared with the mice in the PBS group, the bronchial mucosa of the lung tissue of the mice in the OVA group had different degrees of inflammatory changes, mainly manifested as destruction, shedding and loss of airway epithelial cells; thickening of the airway wall, infiltration of a large number of inflammatory cells around the wall, increased connective tissue, thickening of local alveolar septa, and hyperplasia of airway smooth muscle. The infiltration of inflammatory cells in the MY2024 group of mice was significantly reduced, and the destruction of the airway epithelium was significantly alleviated compared with the OVA group ( Figure 11-13 ). PAS staining showed that compared with the PBS group, the goblet cell proliferation of the lung tissue of the mice in the OVA group increased significantly, and the mucus secretion increased, while the mucus secretion of the MY2024 group decreased significantly compared with the OVA group ( Figure 14-16 MASSON staining showed that compared with the PBS group, the lung tissue of the OVA group mice had obvious collagen deposition, while the collagen deposition in the MY2024 group was significantly improved ( Figure 17-Figure 19 ).

[0062] (3) After 21 days of MY2024 intervention, the expression of marker genes for macrophage activation in the lung tissue of mice (including CD206, Arg1, Fizz1 and Ym1 ) expression was significantly lower than that in the OVA group ( Figure 20-23 The quantitative results are shown in Table 2). The expression of Arg1, a marker of macrophage alternative activation, was significantly lower in the OVA group than in the OVA group, suggesting that the OVA-induced macrophage alternative activation may be improved.

[0063] Table 2. Comparison of relative expression levels of macrophage alternative activation genes in animals of each experimental group

[0064] Fig.24 Oral administration of MY2024 significantly reduced the level of Arg1 protein, a marker gene for alternative activation of macrophages in lung tissue of OVA mouse model ( Fig.24 PBS group 0.09±0.07, OVA group 1.03±0.20, MY2024 group 0.60±0.30).

[0065] In summary, compared with the OVA group, oral administration of the MY2024 strain can significantly improve the asthma symptoms of mice, reduce lung inflammatory cell infiltration, mucus secretion, collagen deposition and pulmonary fibrosis, and reduce excessive macrophage alternative activation in the lungs, indicating that the MY2024 strain of Enterobacter mesenteroides has a good anti-asthmatic effect and is of great significance for the prevention and treatment of asthma.

Claims

1. A strain of Leuconostoc mesenteroides subspecies jonggajibkimchii, the deposit number of which is CGMCC No. 30904, the deposit date is June 7, 2024, and the deposit classification is named Leuconostoc mesenteroides Leuconostoc mesenteroides The preservation unit is the General Microbiology Center of China Microbiological Culture Collection Administration.

2. The strain according to claim 1, characterized in that The sequence of 16S rRNA of the strain is shown as SEQ ID NO.

1.

3. Use of the strain according to claim 1 or 2 in the preparation of a drug for preventing or treating allergic airway inflammation.

4. The use according to claim 3, characterized in that: The allergic airway inflammation is asthma.

5. The use according to claim 4, characterized in that: The asthma is accompanied by an increase in serum IgE and / or Th2 cytokines in the airways.

6. The use according to claim 4, characterized in that: Asthma is associated with inflammatory cell infiltration, goblet cell hyperplasia, mucus production, and collagen deposition within the airways.

7. The use according to claim 4, characterized in that: Asthma is accompanied by an exaggerated alternative activation of pulmonary macrophages.

8. A composition comprising the strain according to claim 1 or 2.

9. The composition according to claim 8, characterized in that The composition further contains a pharmaceutically acceptable carrier and / or excipient.

10. The composition according to claim 9, characterized in that The composition is prepared as capsules, lyophilized powders, suspensions or tablets.

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